Screening of Mutant Lines and Varieties/Hybrids of Tomato (Solanum lycopersicum) for Resistance to the Northern Root-Knot Nematode Meloidogyne hapla

Comments 1: Join the paragraphs.

Response 1: The annotation structure has been revised.

Comments 2: The current nomenclature of the gene is Mi-1.2 (correct this along the entire manuscript)

Response 2: Thank you for your comment. In the entire text of the article, including figures and tables, the designation of the gene is adjusted in accordance with the current nomenclature — Mi-1.2.

Comments 3 General information needs update: Tomato plants carrying the Mi-1.2 gene are resistant to 13 Meloidogyne species. Plants carrying this gene do not display resistance against M. enterolobii and M. hapla.

Reference: Gabriel, M., Kulczynski, S. M., Muniz, M. F., Boiteux, L. S., & Carneiro, R. M. (2020). Reaction of a heterozygous tomato hybrid bearing the Mi‐1.2 gene to 15 Meloidogyne species. Plant pathology, 69(5), 944-952.

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have expanded the Introduction section to include information on the resistance spectrum of Mi-1.2. Data regarding the gene’s lack of efficacy against Meloidogyne hapla and M. enterolobii have been added, along with the appropriate reference:
Gabriel, M. et al. (2020). Plant Pathology, 69(5), 944–952.
This change can be found on pages 2–3, lines 40–61 of the revised manuscript.

Comments 4: he histopathological analysis of the infection process of plants with and without the Mi-1.2 gene was recently described in details

Reference: Gabriel, M., Santos, M. F., Mattos, V. S., Gomes, A. C. M., de Almeida, S. F., Castagnone-Sereno, P., ... & Carneiro, R. M. (2024). Comparative histopathology of virulent and avirulent Meloidogyne javanica populations on susceptible and resistant tomato plants. Frontiers in Plant Science, 15, 1425336.

Response 4: The introduction mentions the latest histopathological study demonstrating differences in the formation of feeding sites in sensitive and resistant plants: Gabriel M. et al. (2024). Frontiers in Plant Science, 15, 1425336. This change can be found on page 3, lines 48–50 of the revised manuscript.

Comments 5: Some words of Latin origin are missing the italics

Response 5: Latin biological names were italicized in all sections of the manuscript, including the title and abstract.

Comments 6: Even though, very interesting the use of Figure 2 would be nice in a review paper (which is not the case).

Response 6: Thank you for the recommendation. Figure 2 has been deleted because its overview character goes beyond the format of the original research report. All data is saved as a description in the "introduction" section.

Comments 7: One thing that makes the results doubtful is the resistant reaction of the cultivar Rio Grande (which is a well-know susceptible cultivar). Is the name of this cultivar correct?

Ответ 7: Название сорта подтверждено: «Рио-Гранде». Тем не менее, мы благодарим вас за ваш комментарий и уточняем в тексте, что по нашей шкале устойчивости он классифицируется как «устойчивый» на основании относительно низкого индекса галлообразования, но не демонстрирует полного иммунитета. В текст статьи добавлено объяснение разницы между полным сопротивлением и умеренным сопротивлением. Это изменение можно найти на стр. 12, строки 422–426 исправленной рукописи.

Comments 1: The introduction outlines the context adequately, but the explanation regarding the genetic basis of resistance could be expanded. The Mi-1 gene is presented as the main marker, yet its limitations under high-temperature conditions or in relation to specific Meloidogyne species are not discussed. This omission affects the depth of the rationale for using this marker exclusively. References to alternative loci or mechanisms would strengthen the foundation of the study.

Response 1: We appreciate this valuable suggestion. In the revised version of the manuscript, the Introduction section has been expanded to include a detailed explanation of the limitations of Mi-1.2-mediated resistance, particularly its temperature sensitivity (loss of efficacy above 28 °C due to the instability of the HSP90–Mi-1.2 complex) and its ineffectiveness against M. hapla and M. enterolobii. This change can be found on pages 2-3, lines 40–61 of the revised manuscript.

In addition, we now reference alternative resistance loci such as Mi-3, Mi-5, and Mi-9, as well as potential contributions from polygenic resistance mechanisms, MAPK signaling cascades, WRKY transcriptional regulation, and hormone-mediated responses (SA, JA, and ET). This change can be found on pages 3-4, lines 87–97 of the revised manuscript.

Comments 2: In the methodology, some important details are missing. The gall index scoring system is referenced but not clearly described, and the number of replicates per treatment is not explicitly stated. These gaps limit the reproducibility of the study. The gel image used to present the SCAR marker results lacks clarity. It is difficult to interpret due to low contrast, and the sample identities are not annotated. This figure could be improved or replaced by a schematic summarizing the presence or absence of the Mi-1 gene in the evaluated genotypes.

Response 2: Thank you for your observation. We revised the Materials and Methods section to clearly describe the gall index (GI) scoring system based on the 0–5 Taylor and Sasser scale. In addition, we clarified the experimental design. This change can be found on page 6, lines 205–209; 224-229 of the revised manuscript. Regarding the gel image, we replaced the figure with a higher-quality version featuring improved contrast and clearly labeled sample identities. This change can be found on Fig. 6, page 11

Comments 3: The results indicate a consistent trend between resistance and maintenance of vitamin C content in fruits. However, the presentation of these data lacks statistical support. No indication is given of variability measures, and the type of statistical test applied for mean comparisons is not specified. Without this information, it is difficult to evaluate the reliability of the observed differences.

Response 3: We appreciate this important remark. The Results section has been revised to include statistical support for the vitamin C data. We now report mean values with standard deviations (mean ± SD) for each group in Figures 4–5, page 10. In addition, a two-way ANOVA was described in more detail to assess the effects of resistance status and nematode infection, followed by Tukey’s HSD post hoc test for group mean comparisons. Significant differences are now clearly indicated in the text. This change can be found on page 9-10 of the revised manuscript.

Comments 4: An important finding is the identification of genotypes that exhibit resistance without the presence of the Mi-1 marker. This observation suggests a more complex or polygenic basis for resistance, but the discussion does not explore this possibility adequately. Greater emphasis on these lines and on the implications for breeding would increase the impact of the study.

Response 4:. Thank you for this valuable comment. In the revised Discussion section, we expanded our interpretation of Mi-1.2-negative but phenotypically resistant genotypes, with particular focus on the cultivar Volgogradets, which exhibited complete immunity (GI = 0) in the absence of the SCAR marker. This case is now discussed as a likely example of Mi-independent resistance, potentially governed by polygenic architecture or alternative signaling mechanisms. We also emphasized the breeding value of such genotypes, highlighting their potential to serve as donors of durable resistance traits. This discussion is supported by relevant literature on alternative resistance loci (Mi-3, Mi-5, Mi-9) and molecular mechanisms such as WRKY-mediated transcriptional regulation, MAPK signaling, reactive oxygen species (ROS) production, and hormone-mediated defense responses. . This change can be found on page 11-13 of the revised manuscript.

Comments 5: The integration between the different methods could also be improved. Currently, the morphological, biochemical, and molecular results are presented as separate layers. A more integrated interpretation would enhance the narrative and better reflect the multidimensional nature of the screening.

Response 5: In the revised Discussion, we have added an integrative section that brings together the morphological (gall index), biochemical (vitamin C content), and molecular (SCAR marker presence) finding . This change can be found on page 13, lines 446–461 of the revised manuscript.